A competitive transcription factor binding mechanism determines the timing of late cell cycle-dependent gene expression.

Transcriptional control is exerted by the antagonistic activities of activator and repressor proteins. In Saccharomyces cerevisiae, transcription factor complexes containing the MADS box protein Mcm1p are key regulators of cell cycle-dependent transcription at both the G2/M and M/G1 transitions. The homeodomain repressor protein Yox1p acts in a complex with Mcm1p to control the timing of gene expression. Here, we show that Yox1p interacts with Mcm1p through a motif located N terminally to its homeodomain. Yox1p functions as a transcriptional repressor by competing with the forkhead transcription activator protein Fkh2p for binding to Mcm1p through protein-protein interactions at promoters of a subset of Mcm1p-regulated genes. Importantly, this competition is not through binding the same DNA site that is commonly observed. Thus, this study describes a different mechanism for determining the timing of cell cycle-dependent gene expression that involves competition between short peptide motifs in repressor and activator proteins for interaction with a common binding partner.

SWR1 chromatin remodeling complex prevents mitotic slippage during spindle position checkpoint arrest.

Faithful chromosome segregation in budding yeast requires correct positioning of the mitotic spindle along the mother to daughter cell polarity axis. When the anaphase spindle is not correctly positioned, a surveillance mechanism, named as the spindle position checkpoint (SPOC), prevents the progression out of mitosis until correct spindle positioning is achieved. How SPOC works on a molecular level is not well understood. Here we performed a genome-wide genetic screen to search for components required for SPOC. We identified the SWR1 chromatin-remodeling complex (SWR1-C) among several novel factors that are essential for SPOC integrity. Cells lacking SWR1-C were able to activate SPOC upon spindle misorientation but underwent mitotic slippage upon prolonged SPOC arrest. This mitotic slippage required the Cdc14-early anaphase release pathway and other factors including the SAGA (Spt-Ada-Gcn5 acetyltransferase) histone acetyltransferase complex, proteasome components and the mitotic cyclin-dependent kinase inhibitor Sic1. Together, our data establish a novel link between SWR1-C chromatin remodeling and robust checkpoint arrest in late anaphase.

Polo kinase controls cell-cycle-dependent transcription by targeting a coactivator protein.

Polo kinases have crucial conserved functions in controlling the eukaryotic cell cycle through orchestrating several events during mitosis. An essential element of cell cycle control is exerted by altering the expression of key regulators. Here we show an important function for the polo kinase Cdc5p in controlling cell-cycle-dependent gene expression that is crucial for the execution of mitosis in the model eukaryote Saccharomyces cerevisiae. In particular, we find that Cdc5p is temporally recruited to promoters of the cell-cycle-regulated CLB2 gene cluster, where it targets the Mcm1p-Fkh2p-Ndd1p transcription factor complex, through direct phosphorylation of the coactivator protein Ndd1p. This phosphorylation event is required for the normal temporal expression of cell-cycle-regulated genes such as CLB2 and SWI5 in G2/M phases. Furthermore, severe defects in cell division occur in the absence of Cdc5p-mediated phosphorylation of Ndd1p. Thus, polo kinase is required for the production of key mitotic regulators, in addition to previously defined roles in controlling other mitotic events.

Cell cycle-regulated transcription through the FHA domain of Fkh2p and the coactivator Ndd1p.

Recent studies in Saccharomyces cerevisiae by using global approaches have significantly enhanced our knowledge of the components involved in the transcriptional regulation of the cell cycle. The Mcm1p-Fkh2p complex, in combination with the coactivator Ndd1p, plays an important role in the cell cycle-dependent expression of the CLB2 gene cluster during the G2 and M phases ([4-7]; see [8-10]for reviews). Fkh2p is phosphorylated in a cell cycle-dependent manner, and peak phosphorylation occurs coincidentally with maximal expression of Mcm1p-Fkh2p-dependent gene expression. However, the mechanism by which this complex is activated in a cell cycle-dependent manner is unknown. Here, we demonstrate that the forkhead-associated (FHA) domain of Fkh2p directs cell cycle-regulated transcription and that the activity of this domain is dependent on the coactivator Ndd1p. Ndd1p was found to be phosphorylated in a cell cycle-dependent manner by Cdc28p-Clb2p, and, importantly, this phosphorylation event promotes interactions between Ndd1p and the FHA domain of Fkh2p. Furthermore, mutation of the FHA domain blocks these phosphorylation-dependent interactions and abolishes transcriptional activity. Our data therefore link the transcriptional activity of the FHA domain with cell cycle-dependent phosphorylation of the coactivator Ndd1p and reveal a mechanism that permits precise temporal activation of the Mcm1p-Fkh2p complex.

Regulation of cell cycle-specific gene expression through cyclin-dependent kinase-mediated phosphorylation of the forkhead transcription factor Fkh2p.

The forkhead transcription factor Fkh2p acts in a DNA-bound complex with Mcm1p and the coactivator Ndd1p to regulate cell cycle-dependent expression of the CLB2 gene cluster in Saccharomyces cerevisiae. Here, we demonstrate that Fkh2p is a target of cyclin-dependent protein kinases and that phosphorylation of Fkh2p promotes interactions between Fkh2p and the coactivator Ndd1p. These phosphorylation-dependent changes in the Fkh2p-Ndd1p complex play an important role in the cell cycle-regulated expression of the CLB2 cluster. Our data therefore identify an important regulatory target for cyclin-dependent kinases in the cell cycle and further our molecular understanding of the key cell cycle regulatory transcription factor Fkh2p.

Mcm1p-induced DNA bending regulates the formation of ternary transcription factor complexes.

The yeast MADS-box transcription factor Mcm1p plays an important regulatory role in several diverse cellular processes. In common with a subset of other MADS-box transcription factors, Mcm1p elicits substantial DNA bending. However, the role of protein-induced bending by MADS-box proteins in eukaryotic gene regulation is not understood. Here, we demonstrate an important role for Mcm1p-mediated DNA bending in determining local promoter architecture and permitting the formation of ternary transcription factor complexes. We constructed mutant mcm1 alleles that are defective in protein-induced bending. Defects in nuclear division, cell growth or viability, transcription, and gene expression were observed in these mutants. We identified one likely cause of the cell growth defects as the aberrant formation of the cell cycle-regulatory Fkh2p-Mcm1p complex. Microarray analysis confirmed the importance of Mcm1p-mediated DNA bending in maintaining correct gene expression profiles and revealed defects in Mcm1p-mediated repression of Ty elements and in the expression of the cell cycle-regulated YFR and CHS1 genes. Thus, we discovered an important role for DNA bending by MADS-box proteins in the formation and function of eukaryotic transcription factor complexes.

Molecular determinants of the cell-cycle regulated Mcm1p-Fkh2p transcription factor complex.

The MADS-box transcription factor Mcm1p and forkhead (FKH) transcription factor Fkh2p act in a DNA-bound complex to regulate cell-cycle dependent expression of the CLB2 cluster in Saccharomyces cerevisiae. Binding of Fkh2p requires prior binding by Mcm1p. Here we have investigated the molecular determinants governing the formation of the Mcm1p- Fkh2p complex. Fkh2p exhibits cooperativity in complex formation with Mcm1p and we have mapped a small region of Fkh2p located immediately upstream of the FKH DNA binding domain that is required for this cooperativity. This region is lacking in the related protein Fkh1p that cannot form ternary complexes with Mcm1p. A second region is identified that inhibits Mcm1p-independent DNA binding by Fkh2p. The spacing between the Mcm1p and Fkh2p binding sites is also a critical determinant for complex formation. We also show that Fkh2p can form ternary complexes with the human counterpart of Mcm1p, serum response factor (SRF). Mutations at analogous positions in Mcm1p, which are known to affect SRF interaction with its partner protein Elk-1, abrogate complex formation with Fkh2p, demonstrating evolutionary conservation of coregulatory protein binding surfaces. Our data therefore provide molecular insights into the mechanisms of Mcm1p- Fkh2p complex formation and more generally aid our understanding of MADS-box protein function.

Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-kappaB-dependent gene transcription in BCG-stimulated macrophages through promotion of p65/p300 binding.

The proinflammatory response of infected macrophages is an important early host defense mechanism against mycobacterial infection. Mycobacteria have been demonstrated to induce proinflammatory gene transcription through the Toll-like receptors, (TLR)2 and TLR 4, which initiate signaling cascades leading to nuclear factor (NF)-kappaB activation. The main transduction pathway responsible for NF-kappaB activation has been established and involves the MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor-6, NF-kappaB-inducing kinase, and inhibitor of kappaB kinase complex. The role of other kinase cascades triggered by mycobacteria in the NF-kappaB activation is less clear. We herein examine the role of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI-3K) cascades in the expression of the bacillus Calmette-Guerin (BCG) mycobacteria-induced NF-kappaB-dependent genes, macrophage-inflammatory protein-2 (MIP-2) and inducible nitric oxide (NO) synthase. Specific pharmacological inhibition of the PI-3K, c-jun-N-terminal kinase (JNK), and to a smaller extent, p38 MAPK but not extracellular-regulated kinase (ERK), suppressed NF-kappaB-dependent reporter gene transcription and MIP-2 and NO secretion in BCG-induced RAW264.7 macrophages. A similar effect was obtained following molecular inhibition of JNK via JNK-interacting protein-1 overexpression. In addition, a kinase-dead mutant of MEK kinase-1, the up-stream regulator of JNK, also proved to be a potent inhibitor of NF-kappaB-reporter activity. The effect of inhibitors was mediated by the down-regulation of NF-kappaB transcription activity and without effecting its nuclear translocation. These data suggest an indirect mechanism of the NF-kappaB regulation by these kinases, probably through p65 phosphorylation and improved binding to the p300 transcription coactivator. The data obtained demonstrate that PI-3K, JNK, and p38 MAPK activation by mycobacteria enhance NF-kappaB-driven gene expression contributing to the proinflammatory macrophage response.

Protein kinase C coordinates histone H3 phosphorylation and acetylation.

The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly.

Lre1 directly inhibits the NDR/Lats kinase Cbk1 at the cell division site in a phosphorylation-dependent manner.

BACKGROUND: The nuclear Dbf2 related (NDR) family of protein kinases play important roles in cell-cycle regulation, apoptosis, cell morphogenesis, and development in a variety of organisms. In budding yeast, the NDR kinase complex composed of Cbk1 and its regulatory subunit, Mob2, have an established role in the control of cell separation/abscission that follows cytokinesis. Whereas the activators of Cbk1-Mob2 have been more extensively described, the mechanisms that restrict or inhibit Cbk1-Mob2 catalytic activity remain largely unknown. RESULTS: We identified the protein Lre1 as a direct inhibitor of Cbk1-Mob2 catalytic activity. We show that Lre1 accumulates at the cell division site in late anaphase and associates with both Mob2 and Cbk1 in vivo and in vitro. Biochemical and functional analysis established that the ability of Lre1 to associate with Cbk1-Mob2 was reduced by mitotic Cdk1 activity and promoted by Cdc14 phosphatase at the end of mitosis. The inhibition of Cbk1-Mob2 by Lre1 was critical to promote the survival of cells lacking the actomyosin driven pathway of cytokinesis. CONCLUSIONS: We established Lre1 as a direct inhibitor of the NDR kinase Cbk1-Mob2, which is regulated in a cell-cycle-dependent manner. We propose that similar inhibitory proteins may also provide fine tuning for the activity of NDR kinases in other organisms.

Protein kinase C regulates late cell cycle-dependent gene expression.

The control of the cell cycle in eukaryotes is exerted in part by the coordinated action of a series of transcription factor complexes. This is exemplified by the Mcm1p-Fkh2p-Ndd1p complex in Saccharomyces cerevisiae, which controls the cyclical expression of the CLB2 cluster of genes at the G(2)/M phase transition. The activity of this complex is positively controlled by cyclin-dependent kinase (CDK) and polo kinases. Here, we demonstrate that the protein kinase Pkc1p works in the opposite manner to inhibit the activity of the Mcm1p-Fkh2p-Ndd1p complex and the expression of its target genes. In particular, Pkc1p causes phosphorylation of the coactivator protein Ndd1p. Reductions in Pkc1p activity and the presence of Pkc1p-insensitive Ndd1p mutant proteins lead to changes in the timing of CLB2 cluster expression and result in associated late cell cycle defects. This study therefore identifies an important role for Pkc1p in controlling the correct temporal expression of genes in the cell cycle.

Transfection of NS0 myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by improving cellular resistance to apoptosis.

Mouse myeloma NS0 cells widely used in hybridoma technology lack the expression of a major stress protein Hsp70 which is the principal component of the basic cellular defense mechanism. These cells rapidly undergo apoptosis at the late-stationary phase of batch culture following nutrient exhaustion. Since Hsp70 was recently demonstrated to protect cells against numerous apoptotic stimuli, the aim of the present study was to examine the protective potential of the protein expression in engineered myeloma NS0 cells and in resulting hybridomas. Myeloma cells were transfected with the hsp70 gene under beta-actin gene promoter. To imitate harmful conditions that hybridoma or myeloma cells often experience when cultivated in large scale for an antibody production, NS0(wt) and NS0(hsp70) cell cultures were maintained without changing the medium for a few days, and the expression of apoptotic markers has been studied. It was found that long-term cultivation induced apoptosis in original cells manifested by typical nuclei fragmentation, DNA ladders and activation of caspase-3. In contrast, in transfected cells under the same conditions the outcome of apoptosis was postponed for 24 hours. Most relevant was that the fusion of transfected myeloma cells with immune splenocytes resulted in twofold hybridomas output compared with wild-type fusion partner. Almost half of the hybridomas continued to be hsp70-positive and maintained higher robustness in culture. The level of monoclonal antibodies production by hybridoma cells obtained with the use of NS0(wt) and NS0(hsp70) was similar, however, the secreted product was better preserved in culture supernatants of Hsp70-positive cells. It is concluded that transfection of mouse myeloma cells with the hsp70 gene can be a novel means to increase hybridoma yield and reduce the sensitivity of myeloma and hybridoma cells to culture conditions insults accompanying monoclonal antibody production.